Polyketides are a group of extremely diverse natural products produced by polyketide synthases (PKSs). These compounds are typically isolated from actinomycetes and have a long and successful history in finding wide ranging applications as antibacterial, antifungal, anticancer and antiparasitic agents as well as immunosuppressants. Combinatorial biosynthesis, in which components of different polyketide biosynthetic systems are combined in a rational or random manner, can assess a wide range of novel polyketide structures and has recently attracted considerable interest as a potentially important and powerful new tool in natural product drug discovery. PKSs typically use simple acyl thioesters as biosynthetic precursors. The supply of precursors for natural and hybrid PKSs determines both amount and type of polyketide product produced in a fermentation process, yet remains a poorly understood process. This project will use a combination of gene disruption and overexpression, isotope incorporation studies, precursor-directed biosynthesis, and enzyme purification and characterization studies to understand both the relative roles of different pathways in providing common precursors for PKSs, and the manner in which these pathways can be manipulated to give access to novel polyketides. The first two specific aims of the project will address formation of methymalonyl CoA (MMCoA) and ethylmalonyl CoA (EMCoA), important precursors used by a wide range of modular type I PKSs. Using the monensin producer Streptomyces cinnamononensis as a host the role of propionyl CoA carboxylase, crotonyl CoA reductase, isobutyryl CoA mutase, and acetoacetyl CoA thiolase (AAT) in providing these precursors will be addressed. In addition, the role of a novel coenzyme B12-dependent mutase (encoded by meaA), which catalyzes a carbon-skeleton rearrangement in a previously unidentified pathway to MMCoA, will be investigated. The importance of this novel pathway relative to established pathways for MMCoA will be determined. The remaining specific aims address pathways responsible for generating acetyl ACP (acyl carrier protein) and butytryl ACP, precursors used to initiate biosynthesis in iterative type II PKSs. The role of 3-ketoacyl ACP synthase III (KSIII), KSIII homologs and AAT with a unique acetyl CoA:ACP transacylase activity, in generating these precursors in vivo will be determined. This research will be carried out using the tetracenomycin producer Streptomyces glaucescens, and the frenolicin producer Streptomyces roseofulvus as hosts. The use of KSIII and KSIII homologs to generate novel acyl ACP starter units for polyketide biosynthesis and the ability of the PKSs to process these starter units will also be investigated.